High frequency (139.5 GHz) electron paramagnetic resonance characterization of Mn(II)-H217O interactions in GDP and GTP forms of p21 ras

Brendan F. Bellew, Christopher J. Halkides, Gary J. Gerfen, Robert G. Griffin, David J. Singel

Research output: Contribution to journalArticlepeer-review

41 Scopus citations


As a molecular switch, the ras protein p21 undergoes structural changes that couple recognition sites on the protein surface to the guanine nucleotide-divalent metal ion binding site. X-ray crystallographic studies of p21 suggest that coordination between threonine-35 and the divalent metal ion plays an important role in these conformational changes. Recent ESEEM studies of p21 in solution, however, place threonine-35 more distant from the metal and were interpreted as weak or indirect coordination of this residue. We report high frequency (139.5 GHz) EPR spectroscopy of p21·Mn(II) complexes of two guanine nucleotides thai probes the link between threonine-35 and the divalent metal ion. By analysis of high-frequency EPR spectra, we determine the number of water molecules in the first coordination sphere of the manganous ion to be four in p21·Mn(II)·GDP, consistent with prior low- frequency EPR and X-ray crystallographic studies. In the complex of p21 with a GTP analog, p21·Mn(II)·GMPPNP, we determine the hydration number to be 2, also consistent with crystal structures. This result rules out indirect coordination of threonine-35 in the solution structure of p21·Mn(II)·GMPPNP, and implicates direct, weak coordination of this residue as suggested by Halkides et al. [(1994) Biochemistry 33,4019]. The 17O hyperfine coupling constant of H217O is determined as 0.25 mT in the GDP form and 0.28 mT in the GTP form. These values are similar to reported values for 17O-enriched aquo ligands and some phosphato ligands in Mn(II) complexes. The high magnetic field strength (4.9 T) employed in these 139.5 GHz EPR measurements leads to a narrowing of the Mn(II) EPR lines that facilitates the determination of 17O hyperfine interactions.

Original languageEnglish (US)
Pages (from-to)12186-12193
Number of pages8
Issue number37
StatePublished - 1996
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry


Dive into the research topics of 'High frequency (139.5 GHz) electron paramagnetic resonance characterization of Mn(II)-H<sub>2</sub><sup>17</sup>O interactions in GDP and GTP forms of p21 ras'. Together they form a unique fingerprint.

Cite this